The present invention relates to microscopic imaging, and more particularly, to simultaneous image scanning at a variety of focal depths for improved automatic focusing during continuous imaging of a microscopic specimen. The invention is advantageous for rapid scanning of an entire microscope slide at high image resolution.
The performance of a scanning cytometry system based on optical microscopy depends both on the scanning instrumentation""s characteristics and on the specimen""s characteristics. The instrumentation performance criteria include: lateral resolution, depth of field, focus range, focus accuracy, focus frequency response, slide scanning rate, photometric sensitivity and dynamic range. Important specimen characteristics include tissue thickness and slide flatness in relationship to the instrument""s objective numeric aperture (NA). Biological specimens do not lie in a single focal plane across a microscope slide which complicates automated scanning of the specimen for image cytometry using typically narrow depth-of-field microscope optics.
Adapting existing scanning systems to optical microscopy often results in a compromise between scanning speed and image resolution. For example, web inspection systems with time-delay-and-integrate (TDI) sensors are used in automated high speed visual inspection systems to detect and identify defects on continuously moving objects. The object in the field of view of the sensor is assumed to be flat and a 2D image is formed by synchronizing the line frequency of the camera to the web movement. When the web or conveyor is not flat, image quality can be degraded. Therefore, the depth of field is usually made large and carefully adjusted to include the largest variations in axial position. In these applications resolution is not a limiting factor and can be sacrificed to obtain large depths of field and avoid the need for an autofocus system. When web inspection techniques are applied to optical microscopy, as in the macroscopic implementations, large depths of field were ensured by the use of low NA objectives in order to avoid the need for autofocus. This is appropriate in low resolution imaging over limited areas, but requires depth of fields of on the order of 10 microns (xcexcm) or more over the length of a standard microscope slide. For higher resolution imaging, the depth of field is limited to about 1.0 xcexcm and refocusing at each field of view is required to improve image quality.
Another exemplary scanning system is optical profilometry. In optical profilometry, a point source of light is imaged on the specimen. Best focus is determined essentially by minimum point size and a focus error detection system is used to measure surface topography. A number of different methods are used to produce an error signal proportional to height variations from minimum point size and this error signal is used to servo control best focus. The height variations are then recorded as lens positions during scanning. The data show horizontal resolutions of 1 to 0.01 xcexcm and vertical resolutions of 0.5 to 0.001 xcexcm. However, the surface slopes of the specimen have a significant influence on the measurement accuracy. One Profilometer follows the specimen surface very well at repositioning frequency of 100 hertz (Hz) with specimen height variations up to 0.2 xcexcm. In open loop mode, the objective lens is fixed and the surface height is derived directly from the focus error. Open loop mode increases the measurement frequency, but a servo is required when the axial range is large such as with biological specimens. Since data is gathered one point at a time, this method is slow and apparently has not been applied to semitransparent specimens (like tissues) that can reflect from multiple surfaces.
Accordingly, there exists a definite need for an automatic microscopic scanning system that can rapidly scan, at high resolution, an entire microscope slide containing a biological specimen. The present invention satisfies these needs and provides further related advantages.
The present invention is embodied in an imaging apparatus, and related method, for fully automatic, high-speed, high-resolution image cytometry. The imaging apparatus updates the focus many times across a conventional microscope field of view while moving the stage in a continuous motion at a constant velocity. The improved focus accuracy and allows performance of high resolution scanning image cytometry at high speeds and analysis of an entire microscope slide in several minutes.
Other features and advantages of the present invention should become apparent from the following description of the preferred embodiments, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention.